Coating plates
Hanqin Li, Oriol Busquets, Steven Poser, Dirk Hockemeyer, Frank Soldner
Abstract
This protocol describes the process of coating plates using either VTN, Matrigel or Geltrex for use in culturing of feeder-free human pluripotent stem cells (hPSCs)
Protocol overview
Coating plates
A. VTN
B. Matrigel
C. Geltrex
General Notes:
- Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.
Steps
A. VTN
Thaw aliquoted VTN On ice. Each 6-well plate needs 60 µl VTN.
For each well of 6-well plate, dilute 10 µl VTN in 1 ml pre-chilled DPBS.
Add the diluted VTN to each well, shake and tilt the plate so the VTN solution covers the entire well.
Incubate in a 37°C incubator for 1h 0m 0s
B. Matrigel
Thaw sufficient Matrigel On ice to coat plates (100 µl Matrigel/6-well plate)
Pipet 100 µl Matrigel into 6 ml cold DMEM/F12
Quickly transfer 1 ml/well of diluted Matrigel to 6-well plate.
Distribute Matrigel evenly across the surface.
Incubate plates at 37°C for 1h 0m 0s
Remove excess Matrigel and wash with DPBS.
Immediately plate cells at the desired density.
C. Geltrex
Thaw sufficient Geltrex on ice to coat plates (120 µl/6-well plate)
Dilute Geltrex 1:50 with cold DMEM/F12 (120 µl Geltrex into 6 ml DMEM/F12)
Quickly transfer 1 ml/well of diluted Geltrex to 6-well plate.
Distribute Geltrex evenly across the surface.
Incubate plates at 37°C for 1h 0m 0s
Remove excess liquid from the Geltrex coated plate.
Immediately plate cells at the desired density.
